SK13232001A3 - Novel lhrh antagonists with improved solubility characteristics - Google Patents

Abstract

The invention relates to peptides which contain N-methylated amino acid building blocks and are provided with improved water solubility. Medicaments containing the inventive peptides can be used for the treatment of hormone-dependent tumours and hormone-influenced, non-malignant diseases.

Description

Technical field

The invention relates to LHRH-antagonists with improved solubility properties, to methods of making these compounds, to medicaments in which they are contained, as well as to the use of medicaments for the treatment of hormone-dependent tumors and malignant diseases affected by hormones such as benign prostatic hyperplasia (BPH); endometriosis.

BACKGROUND OF THE INVENTION

The nomenclature used to define the peptides agrees with the nomenclature explained by the IUPAC-IUB-Commission on the Biochemical Nomenclature (European J. Biochem. 1984, 138, 9-37), where, in accordance with the usual notion, amino groups at the N-terminal end appear in the left and carboxyl groups at the C-terminal end appear in the right-hand direction. LH-RH antagonists as peptides of the invention include naturally occurring and synthetic amino acids, the first including Ala, Val, Leu, Ile, Ser, ThrLys, Arg, Asp, Asn, Glu, Gln, Gys, Cys, Met, Phe, Tyr , Pro, Trp and His. The abbreviations of the individual amino acid residues are derived from the trivial amino acid names and are Ala = alanine, Arg = arginine, Gly = glycine, Leu = leucine, Lys = lysine, Pal (3) = 3- (3-pyridyl) alanine, Nal (2) =

3- (2-naphthyl) alanine, Phe = phenylalanine, Cpa = 4-chlorophenylalanine, Pro = proline, Ser = serine, Thr = threonine, Trp = tryptophan, Tyr = tyrosine and Sar = sarcosine. All amino acids described herein are of L-series unless otherwise indicated. For example, D-Nal (2) stands for 3- (2-naphthyl) -D-alanine and Ser stands for L-serine. Substitutions on the ε-amino group in the lysine side chain are represented by the expression after Lys in parenthesis, optionally in the form of an abbreviation.

Other abbreviations used are:

Ac acetyl

Atz 3-amino-1,2,4-triazole-5-carbonyl

B 4- (4-amidinophenyl) amino-1,4-dioxobutyl

Boe tert-butyloxycarbonyl

Bop benzotriazole-1-oxy-tris- (dimethylamino) -phosphoniumhexafluorophosphate

DCC dicyclohexylcarbodiimide

DCM dichloromethane

Ddz dimethoxyphenyldimethylmethyleneoxycarbonyl (dimethoxymethyl-Z)

DiC diisopropylcarbodiimide

DIPEA N, N-diisopropylethylamine

DMF dimethylformamide

Fmoc fluorenylmethyloxycarbonyl

HF hydrofluoric acid

HOBt 1-hydroxybenzotriazole

HPLC high pressure liquid chromatography

Me methyl

TFA trifluoroacetic acid

Z - benzyloxycarbonyl

Hci homocitrulín

Cpa 4-chlorophenylalanine

The peptides of the present invention are analogs of a lutenizing hormone-influencing hormone (LH-RH) having the following structure:

_{p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2,} (LH-RH, gonadorelin)

For over 20 years, scientists have been searching for selectively potent LH-RH-decapeptide antagonists [M. Karten and J. Riviera, Endocrine Reviews 7, 44-66 / 1986)]. A great interest in such antagonists lies in their benefit in the fields of endocrinology, gynecology, tarchitis prevention and cancer. A large number of compounds were produced as potential LH-RH-antagonists. The most interesting compounds that have been found hitherto are those whose structures represent a modification of the LH-RH structure.

The first series of potent antagonists was obtained by introducing aromatic amino acid residues at positions 1,2,3 and 6 or 2,3 and 6. The usual notation of compounds is as follows: first, the amino acids that entered the LH-RH peptide chain at the site of the originally present amino acids are reported. and the positions at which the exchange occurred are denoted by numbers denoted as superscript. Furthermore, the following designation LH-RH indicates that these are LH-RH analogues that have been exchanged.

Known antagonists are:

[Ac-D-Cpa ¹ ' ² , D-Trp ³ ' ⁶ ] LH-RH (D, H. Coy et al., In: Gross E. and Meienhofer, J. (Eds) peptides; Proceedings of the 6th American Peptide Symposium, pp. 775-779, Pierce Chem., Co. Rockville

III. (1979):

[Ac-Pro ¹ , D-Cpa ² , D-Arg ⁶ ] LH-RH (ORG-30276) (HDCoy, et al. Endocrinology 100, 1445, 1982), and [Ac-D-Na (2) 1, D-Phe (4-F) ² , D-Trp ³ , D-Arg ⁶ ] LH-RH / ORF 18260 (JE Rivier et al .: Vickery BH Nestor, Jr. JJ Hafex,

E. S. E. (Eds). LH-RH and its Analogs. with. 11-2 MPT Press, Lancaster, UK 1984).

Other potent LH-RH-antagonists are described in WO 92/19651, WO 94/19370, WO 92/17025, WO 94/13313,

US-A 5 300 492, US-A 5 140 009, EP 0 413 209 A1 and DE 195 44 212 A1.

The latter disclose compounds having a building block modified with ornithine or lysine at the 6-position, which correspond to the following formula:

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-Xxx ⁶ -Leu ⁷ -Arg ⁸ q η n

Pro-D-Ala-NH ₂ , wherein D-Xxx is an amino acid group of formula (VI) —HN — CH — CO-

I (CH ₂ ) _n

I (VI)

NH

I

CO-R

LH-rH-antagonists are further described in WO 97/19953 a

EP-A-0 326 090.

Other known antagonists are antarelix, ganirelix and cetro relix.

antarelix

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-hArg (Et) 2 ⁶ -Leu ⁷ -hArg (Et) 2 ^{6-to 9} -D-Ala ¹⁰ -NH ₂

cetrorelix

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -Tyr ⁵ -D-Cit ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂

It is an object of the present invention to provide novel LH-RH antagonists which exhibit enhanced enzymatic stability and significantly improved aqueous solubility.

SUMMARY OF THE INVENTION

This task is solved by compounds of the following formula (I)

A-Xxx ¹ -XXX ² -XXX -XXX ^{3 4 5} -XXX -XXX -XXX ^{6 7 8} -XXX -XXX ⁹ -XXX θ ¹ -NH ₂ wherein

A acetyl or 3- (4-fluorophenyl) propionyl group,

Xxx ¹ D-Nal (1) or D-Nal (2)

Xxx ² -Xxx ³ D-Cpa-D-Pal (3) or single bond,

Xxx ⁴ Ser

Xxx ⁵ N-Me-Tyr

Xxx ⁶ D-Cit, D-Hci or D-amino acid group of formula (II)

NH.

(CH ₂ ) "

R ¹ wherein n is 3 or 4, wherein R ¹ is a group of formula III

- (CH ₂ ) _p -CO-NR ² R ³ (III) wherein p is an integer from 1 to 4, R ² is hydrogen or alkyl and R is unsubstituted or substituted aryl or heteroaryl, or R ¹ represents

3-amino-1,2,4-triazole-5-carbonyl or R ¹ represents a ring of formula (IV)

R ⁴ /

wherein q is 1 or 2, R 6 is hydrogen or alkyl, R ⁵ is hydrogen or alkyl, and X is oxygen or sulfur,

Ί

Ύχχ / Leu or No,

Xxx ⁶ Arg or Lys (iPr)

Xxx ⁹ Pro a

Xxx ¹⁰ Ala or Ser, and their salts with pharmaceutically acceptable acids, in particular acetates, embonates and trifluoroacetates.

Preferred compounds of the invention are those wherein Xxx ⁶ is D- [eN- (imidazolidin-2-on-4-yl) -formyl] -Lys,

D- (3-amino-1,2,4-triazole-3-carbonyl) -Lys, abbreviated to D-Lys (Atz) or D- [e-N '- (4-amidinophenyl) amino-1,4-dioxobutyl ] -Lys, abbreviated as D-Lys (B).

Other particularly preferred compounds of the invention are:

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂ ,

Ac-D-Na (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (Atz) ⁶ -Leu ⁷ -Arg ⁸ - For ⁹ - D-Ala ¹⁰ -NH ₂

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (B) ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ - D-Ala ¹⁰ -NH ₂

Ac-D-Na (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -Lys (B) ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ -D- Ala ¹⁰ -NH ₂ ,

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Lys (iPr) ⁸ -For ⁹ - Ala ¹⁰ -NH ₂ ,

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Lys (iPr) ⁸ Dec ⁹ -Sar ^1θ -ΝΗ ₂ ,

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Arg ⁸ -Pro ⁹ - Ser ¹⁰ - NH ₂ ,

3- (4-fluorophenyl) -propionyl-D-Nal (1) ¹ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (Atz) ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ -Ala ¹⁰ -NH ₂ as well as their salts with the aforementioned pharmaceutically acceptable acids.

The compounds of the invention may be used to treat hormone-mediated tumors, particularly prostate cancer or breast cancer, as well as benign indications whose treatment requires LH-RH-hormone suppression. For this purpose, they are mixed with customary carriers and excipients and made up as medicaments.

The synthesis of the compounds of formula (I) is carried out either by classical fragment condensation or by solid phase synthesis according to Merrifield and successive construction using D-lysine acylated already in the side chain with a carboxylic acid of formula R 1 -COOH as well as by decapeptide building with the corresponding carboxylic acids by coupling the amide in the side chain of D-lysine®. Accordingly, the introduction of the R ¹ -CO- group can be carried out at three different process stages: before condensation of individual building blocks to the peptide, after lysine or ornithine has been introduced into the peptide chain, but before condensation of the next building block or after all building blocks have been condensed.

Compounds of formula (I) are synthesized by known methods such as pure solid phase technique, partially solid phase technique (so-called fragment condensation) or classical dissolution coupling (see M. Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, 1984).

Solid phase synthesis methods are described, for example, in the Solid Phase Peptide Synthesis J.M. Stewart and J.D. Pierce Chem. Compagny, Rockford, III, 1984 and in G.

Barany and R.B. Merrifield The Peptides, Ch. 1, pp. 1-285, 1979. Academic Press Inc, Classical Dissolution Syntheses are described extensively in Methoden der organischen Chemie (Houben-Weyl), Synthese von Peptiden E. Wunsch (ed.) 1974, Georg Thieme Verlag, Stuttgart, Germany.

The stepwise construction is carried out, for example, by first covalently bonding an amino acid with a terminal carboxyl group whose amino group is in position and protected to an insoluble carrier which is customary for this purpose, the α-amino acid protecting group is cleaved, to the free amino group thus obtained. binds the nearest protected amino acid through its carboxy group, and in this way, the other amino acids of the peptide to be synthesized are added in the correct order step by step, and after all amino acids have been attached, the finished peptide is cleaved from the carrier and optionally further side protecting groups present. Step condensation is performed by synthesis from the corresponding, normally protected, amino acids in a conventional manner.

The interconnection of the individual amino acids is carried out by methods which are customary for this purpose, in particular:

symmetric anhydride method in the presence of dicyclohexylcarbodiimide carbodiimide method generally carbodiimide hydroxybenzotriazole method (see: The Peptides, Volume 2, Ed. E. Gross and J. Meienhofer).

In the coupling of the fragments, preferably the racemization of the ongoing azide coupling or DCC-1-hydroxybenzotriazole method or DCC-3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine method is preferably used. Activated fragment esters may also be used.

Well-activated N-protected amino acid esters, such as the N-hydroxysuccinimide ester or 2,4,5-trichlorophenyl ester, are particularly suitable for the step condensation of amino acids. Aminolysis can be very well catalyzed by N-hydroxy compounds having approximately acetic acidity, such as 1-hydroxybenzotriazole.

Dehydrogenating groups such as the benzyloxycarbonyl radical (= Z-radical) or weakly acidic leaving groups are suitable as amino protecting groups. Suitable protecting groups for the amino groups in position a include: tertiary butyloxycarbonyl, fluorenylmethyloxycarbonyl, carbobenzoxy or carbobenzthio groups (optionally now with p-bromo or or p-nitrobenzyl radical), trifluoroacetyl, trifluoroacetyl, o-trifluoroethyl group, trifluoroacetyl group a group, a trityl group, a p-toluenesulfonyl group, a benzyl group, a benzyl radical (p-bromo or p-nitrobenzyl) substituted in the benzene ring and an α-phenylethyl radical. For this purpose, reference is made to Jesse P. Greenstein and Milton Winitz: Chemistry of Amino Acids, New York 1961, John Wiley and Sons, Inc. Volume 2, eg p. 883 et seq., Principles of Peptide Synthesis, Springer Verlag 1984,

Solid Phase Peptide Synthesis, JM Stewart and JD Young, Pierce Chem. Company, Rockford, III. 1984, G. Baranyand, RB Merrifield The Peptides. Ch. 1. p. 1-285 (1979), Academic Press Inc. as well as The Peptides, Volume 2., Ed. E. Gross and J. Maienhofer, Academic Press. New York. The protective sku11 pin are suitable in principle for the protection of further functional side groups (OH groups, _NH2 group) of the corresponding acid.

The hydroxyl groups present (serine, threonine) are preferably protected with benzyl or similar groups. The other amino groups which are not in the α position (for example, the amino groups in the w position, the guanidine group of arginine) are preferably orthogonally protected.

The amino acid building blocks can be other than lysine modified by R ¹ -CO- or ornithine, commonly buy. The possible process for the production of the last compounds is as follows:

The 1-α-carboxylic acid group is amidated

2. the ε-amino group is protected with Z,

The 3 .alpha.-amino group is protected with a Boc group so that selectivity is created with respect to the later cleavage of the amino protecting groups.

4. The Z group on the ε-amino group is cleaved off

5. The desired R ⁴ -CO- group is introduced at the ε-amino group.

6. The Boc group on the α-amino group is cleaved off

7. The Z-group is introduced on the α-amino group.

For the introduction of the group R ¹ -CO-, essentially the same methods as described above for coupling amino acids are possible. However, condensation using carbodiimide, for example 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 1-hydroxybenzotriazole, is particularly preferred.

The reaction for coupling the amino acids is carried out in an indifferent solvent or suspending agent which is customary for this purpose (e.g. dichloromethane), whereby dimethylformamide may optionally be added to improve solubility.

Possible synthetic carrier materials are insoluble polymers, for example, polystyrene resin in the form of beads, swellable in an organic solvent (for example, a copolymer of polystyrene and 1% divinylbenzene). Construction of the protected decapeptidamide on methylbenzhydrylamide resin (MBHA resin, i.e. polystyrene resin with methylbenzhydrylamide groups), which provides the desired C-terminal function of the peptide amide after cleavage of HF from the support may be carried out according to the following scheme:

Diagram of the diagram

Peptide synthesis protocol degree of function solvent / reagent (v / v) time

1 washing methanol 2x2 min 2 washing DCM 3x3 min 3 secession DMC / TFA (1: 1) 1 x 30 min 4 washing isopropanol 2x2 min 5 washing methanol 2x2 min 6 washing DCM 2x3 min 7 neutralization CM / DIPEA 3x5 min 8 washing methanol 2x2 min 9 washing DCM 3x3 min 10 STOP addition of Boc-As In DCM + DIC + HOBt 11 coupling DCM, optionally DCM / DCF about 90 min 12 washing methanol 3x2 min 13 washing DCM 2x3 min

Na-Boc protected amino acids are coupled usually in triple excess in the presence of diisopropylcarbodiimide and 1-hydroxybenzotriazole (HOBt) in CH ₂ Cl ₂ / DMF for 90 minutes and the Boc-protecting group is cleaved by treatment with 50% trifluoroacetic acid (TFA) for 0.5 hour. in CH ₂ C1 _2nd The chloroanil test according to Christesen and Kaiser ninhydrin test can serve to control the complete reaction. The residues of the free amino functions are blocked by acetylation in a five fold excess of acetylimidazole in CH ₂ C1 _2nd The sequence of the reaction steps of peptide-resin construction follows from the scheme. To cleave the peptide bound to the resin, the present final product of the synthesis of the solid phase is dried in vacuo over P ₂ 0 _5, and treated with 500 fold excess of HF / anisole 10: 1 v: v for 60 minutes at 0 ° C.

After distillation of HF and anisole in vacuo, the peptidamides precipitate by mixing with anhydrous ethyl ether as a white solid, separation from the co-precipitated polymeric carrier is performed by washing with 50% aqueous acetic acid. By carefully concentrating the solutions acidified with acetic acid in vacuo, the peptides can be obtained as highly viscous oils, which, upon addition of absolute ether, are cold to white solids.

Further purification is carried out by routine preparative high pressure liquid chromatography (HPLC) methods.

Conversion of peptides to their addition salts can be accomplished by reaction with peptides in a known manner. Conversely, the free peptides can be obtained by reacting their acid addition salts with bases. Peptidembonates can be obtained by reacting salts of trifluoroacetic acid (TFA-salts) of the peptide with free embonic acid (pamoic acid) or the corresponding disodium embonic acid salt. To this end, the peptide salt in aqueous solution is supplemented with a solution of the disodium salt of embonate acid in a polar aprotic medium, preferably dimethylacetamide, and the pale yellow precipitate formed is isolated.

DETAILED DESCRIPTION OF THE INVENTION

The following examples serve to illustrate the invention without limiting the invention.

Example 1

Ac-D-Nal (2) 4-D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr 4 -D-Hci ⁸ -Nle ⁷ -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂

The synthesis was carried out according to the solid phase diagram (Peptide-Synthese, p. 11) with DIC coupling (HOBt, starting from 3.3 g of MBHA resin (cycle density 1.08 mmol / g).) After cleavage of HF from the polymeric carrier. 3.4 g of crude peptide was obtained, which was purified by standard preparative HPLC, followed by freeze-drying, to obtain 1.43 g of HPCL-unitary product of formula C72, H96, N17, 014, Cl with a correct FAB mass spectrum: 1458 7 (M + H ⁺ ) (calcd. 1457.7), and the corresponding 1 H-NMR spectrum.

¹ H-NMR (500 MHz, D ₂ O / DMSO-d 6, δ in ppm)

8.7 to 7.2, several m, arom-H incompletely replaced NH; 6.92 and 6.58, 2d, 2x2H, aroma. H p. Cl-Phe, 5.2-3.5, several m,

Ca. H a. Alif. H, 3.2-2.6, several m aromatics. Cp-H, 2.1 to 0.7, several m, remaining aliph. H; 1.70, s, 3H, acetyl, 1.20, d, 3H, Cp-H Leu.

Example 2

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (B) ⁶ -Leu ⁷ -Lys (iPr) ^8- For ⁹ -D-Ala ¹⁰ -NH ₂

The synthesis was carried out according to the solid phase diagram scheme (Peptidsynthese-Protokoll, p. 11) with DIC / HOBt coupling, starting from 4.0 g MBHA resin (cycle density 1.11 mmol / g). After cleavage of HF from the polymeric support, 4.87 g of crude peptide was obtained, which was purified by standard preparative HPLC. Following freeze-drying, 0.93 g of HPLC-unitary product was obtained, which was reacted with 4-amidinophenylamino-4-oxobutyric acid in the presence of BOP as coupling reagent to give the title compound. After re-purification by HPLC, 148 mg of the target compound with the general formula C85, H112, N17, 015, Cl with ESI correction mass spectrum: 1647.6 (M + H ⁺ ) (calcd. 1645.8) and the corresponding H-NMR spectra were obtained. .

1 H-NMR (500 MHz, DSMO-dg, δ in ppm)

10.4 s, 1 Ha. 9.13 s, 2H and 8.94, s. 2H, NH's of 4-amidinoaniline 8.6-7.35, several m. flavorings. H and NH, 7.22 and 7.18, 2d, 4 H aroma. H (pCl) Phe. 6.95 and 6.58, -2d, 4H, aroma. H Tyr; 5.2 to

3.5, several m, Ca-H and aliph. H; 3.3 to 2.4, several m, β-Η and N-CHg 2.1 to 1.1, several m, aliph. H, 1.68, s, 3H, acetyl; 1.20, d, 3H, [beta] - [beta] - [alpha] -β; 0.83, dd, 6H, C6-H-Leu.

Example 3

Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (B) ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ - D-Ala ¹⁰ -NH ₂

The synthesis was carried out according to the solid phase diagram scheme (Peptidsynthese-Protokoll, p. 11) with DIC / HOBt coupling, starting from 4.0 g of MBHA resin (cycle density 0.97 mmol / g). After cleavage of HF from the polymeric carrier, 4.0 g of crude peptide was obtained, which was purified by standard preparative HPLC. After subsequent freeze-drying, it was obtained

1.39 g of an HPLC-unitary product which was reacted with 4-amidinophenylamino-4-oxobutyric acid in the presence of BOP as coupling agent to the desired compound. After re-purification by HPLC, 440 mg of the target compound with the general formula C82, H106, N19, Cl was obtained with a correct ESI mass spectrum: 1632.7 (M + H ⁺ ) (calculated 1632.7) and the corresponding H-NMR spectrum.

¹ H-NMR (500 MHz, DSMO-dg, δ in ppm)

10.4, s, 1H and 9.15, s. 2H, and 9.0, s, 2H, NH's of 4-amidinoaniline; 8.60, m, 2H, aroma, H; 8.3 to 7.2, several m, aroma.

H and NH; 7.27 and 7.20, 2d, 4H. flavorings. H (pCl) Phe, 6.96 and 6.60, 2d 4H, aroma. H Tyr; 5.2-3.5, several m, Ca-H and aliph. H, 3.2 to 2.4, several m, cp-H, and N-CH ₃ , 2.1 to 1.1, several m, aliph. H, 1.74, s, 3H, acetyl; 1.20, d, 3H, Cp-H Ala; 0.85, dd, 6H, C1-H-Leu.

Example 4

Ac-D-Nal (2) ¹ -D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Lys (iPr) ⁸ -For ⁹ - Ala ¹⁰ -NH ₂

The synthesis was carried out according to the solid phase diagram scheme (Peptidsynthese-Protokoll, p. 11) with DIC / HOBt coupling, starting from 2.50 g of MBHA resin (cycle density 1.08 mmol / g). After HF was cleaved from the polymeric carrier, 2.78 g of crude peptide, which was purified by standard preparative HPLC. Following freeze-drying, 400 mg of HPLC-unit product of the general formula C75, H106 N15, 014, Cl with correct ESI mass spectrum was obtained: 1472.6 (M + H ⁺ ) (calc. 1471.7) and the corresponding H 2 -NMR spectrum.

¹ H-NMR (500 MHz, D ₂ O / DSMO-dg, δ In ppm)

8.62, m, 2H, 8.30, m, 2H, 7.80, m, 4H, 7.66, s, 1H, 7.47, m, 2H, 7.36, d, 1H. flavorings. H, 7.25 and 7.20, 2 d, 4H, aroma. H (pCl) Phe, 6.96 and 6.63, 2 d, 4H, aroma. H Tyr, 5.10-4.0, several m, Ca-H and aliph. H, 3.75-2.65, several m. cp-H a N-CH ₃ ; 2.1 to 1.05, several, m. aliph. H; 1,74, s,

3H, acetyl; 1.23, d, 3H, [beta] -H-Ala; 1.20, m, CH ₃ , isoprop.,

0.8, m, 3H, CS-H-Nle.

Example 5

Ac-D-Nal (2) ¹ D-Ca ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Lys (iPr) ⁸ Dec ⁹ -Sar ¹⁰ -NH ₂

The synthesis was carried out according to the solid phase diagram scheme (Peptidsynthese-Protokoll, p. 11) with DIC / HOBt coupling, starting from 2.50 g MBHA resin (cycle density 1.08 mmol / g). After cleavage of HF from the polymeric support, 2.74 g of crude peptide was obtained, which was purified by standard preparative HPLC. Following freeze-drying, 840 mg of HPLC-unit product of the general formula C75, H102, N15, 014, Cl with a correct ESI mass spectrum was obtained: (M + H ⁺ ) (calcd. 1471.7) and the corresponding H · * · -NMR spectrum.

¹ H-NMR (500 MHz, D ₂ O / DSMO-d 6, 6 V ppm)

8.6, m, 2H, 8.3, m, 2H, 7.85, m, 2H, 7.8, m; 2H, 7.65, s, 1H, 7.46, m, 2H, 7.35, d; 1H, aroma. H; 7.23 and 7.17, 2d, 4H, aroma. H (pCl) Phe; 3.75 to 2.6, several m, β-Η and N-CH ₃ ; 2.2 to 1.5, several m, aliph. H, 1.70, s, 3H, acetyl, 1.23, d, 3H, CpAla; 1.20, m CH ₃ isoprop: 0.8, m, 3H, cpNle.

Example 6

3- (4-fluorophenyl) -propionyl-D-Nai (1) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (Atz) ⁶ -Leu ⁷ -Arg ⁸ -Pr ⁹ -D-Ala ¹⁰ - NH ₂

The synthesis was performed according to the solid phase diagram scheme (Peptidsynthese-Protokoll, p. 11) with DIC / HOBt coupling, starting from 9.2 g MBHA resin (cycle density 1.08 mmol / g). After cleavage of HF from the polymeric carrier, 5.8 g of crude peptide was obtained, which was purified by standard preparative HPLC. Subsequent freeze-drying gave 2.0 g of HPLC-unitary unsubstituted octapeptide, from which 0.4 mmol was reacted with 0.5 mmol of 3-amino-1,2,4-triazole-5-carboxylic acid in the presence of PyBOP as coupling agent to 790 mg of the crude product of the title compound. After re-purification by HPLC, 200 mg of cannon compound of formula C64, H86, N17, 012, F with a correct FAB mass spectrum was obtained: 1304.6 (M + H ⁺ ) (calc. 1303.6).

8, 14, m, 1H, 7.90, m, IH, 7.80, m, IH, 7.50, m, 2H, 7.35, m, 2H, 7.0, m, 6H, 7, 63, m, 2H, aromatic. H, 5.0, m, 1H, 4.83, m, 2H, 4.41, ra, 1H, 4.30-4.05, several m, 4H, Ca-H; 3.66 to 2.25, s, N-Me; 2.05 to 1.1, several m, aliph. and aromatics. H side chains; 2.95, s, and 2.75, s, N-Me, 2.05-1.1, several m, aliph. H; 1.20, d, [beta] -H-Ala; 0.75, m, 6H, C 5 -H Leu.

The compounds of the formula I according to the invention have been investigated for their receptor binding. The method was based closely on that described by Beckers et al., Eur. J. Biochem. 231: 535-643 (1995). The Cetrorelix obtained according to the above synthesis was iodinated [ ¹²⁵ J] (Amersham, specific activity 80.5 Bq / fmol using an iodine gene reagent (Pierce). The reaction mixture was purified by reverse-phase high performance liquid chromatography, with mono-iodinated cetrorelix then about 80% of [ ¹²⁵ J] -cetrorelix and the unlabeled compound of the invention were suitable for specific receptor association

The compounds of the invention can be tested by the following methods 1 and 2 for their effect in vitro, wherein the binding affinities were determined by assaying [ ¹²⁵ µ] -centrorelix binding (method 1) and functional activity with triptorelin as an agonist stimulus (method 2).

Method 1

Beckers Receptor Binding Assay, Marheineke, K., Reiländer, H., Hilgard P (1995) Selection and characterization of the mammalian cell lines with stable overexpression of human pituitary receptors for gonadoliberin (GnRH) Eur.J. Biochem. 231: 535-543.

To examine receptor binding, cetrorelix was iodinated using an iodine gene reagent (Pierce) with [ ¹²⁵ J] (Amersham; 80.5 Bq / fmol specific activity). The reaction mixture was purified by reverse phase high performance liquid chromatography to yield mono-iodinated cetrorelix without unlabeled peptide. About 80% of [ ¹²⁵ J] cetrorelix was suitable for spherical association.

The receptor binding assay was performed with intact cells under physiological conditions as described (Beckers et al. 1995). Subconfluent cultures of stably transfected LTK cells that express the human LHRH-receptor were separated by incubation in NaCl / Pi. (137 mM NaCl, 2.7 M KCl, 8.1 mM Na ₂ HPO ₄ , 11.47 mM KH ₂ PO ₄ 1 µM EDTA and were collected by centrifugation. The cell pellet was resuspended in binding buffer (DMEM without H ₂ CO ₃ , with 4.5 g / l glucose, 10 mM hepes pH 7.5, 0.5%, 0.5% (w / v) BSA, 1 g / 1 bacitracin, 0.1 g / l SBD1, 0.1% (w / v) NaN ₃ ). In the wash assay, 0.25x10 ⁶ cells / 100 μΐ were incubated with about 225 µM [ ¹²⁵ J] -cetrorelix (specific activity 5-10x10 ⁵ dpm / pmol and various concentrations of unlabeled compound of the invention as a competitor). the media was stacked in 400 μΐ test tubes above 200 μΐ 84% by volume of silicone oil (Merck Type 550) / 16% by volume of paraffin oil After incubation for 1 hour at 37 ° C with slow, continuous shaking, cells were separated. -minute centrifugation at 9000 rpm (Rotortyp HTA 13.8; Heraeus Sepatec, Osterode, Germany) from the incubation medium.The tube tips containing the cell pellet were cut off, and the cell pellet and overlap were finally analyzed by τ-radiation indication. The amount of non-specific bound cells was determined to exclude unlabeled cetrorelix at a final concentration of 1 μΜ and was typically <10% of all bound cells. ch data was performed with EBDA / ligand analysis program (Biosoft V3, o).

Method 2

Functional assay to determine antagonist activity

The assay was performed with some modifications as described in Beckers, T. Reiländer, H. Hilgard, P. (1997) Characterization of gonadotropin-relapsing hormone analogs based on a sensitive cellular luciferase reporter gene assay analyte. Biochem. 251, 17-23 (Beckers et al.

1997). 10,000 cells per well that express the human LHRH receptor and luciferase gene transporter were cultured for 24 hours in microtiter plates using DMEM with additives and 1% (v: v). The cells were then stimulated for 6 hours with 1 nM [D-Trp] LHRH. The antagonist compounds of the invention were added prior to stimulation and the cells were finally lysed to quantitate cellular Luc activity. IC ₅₀ -values were cut from dose-effect curves using a non-linear regression analysis using a Hill model (Program EDX 2.0 C. Grunwald, Arzneimittelwerk, Dresden).

The quantification of Luc activity was essentially performed as described (Promega Technical Bulletin # 101/161) using the current luciferase assay system (Promega E4030) in duplicate. By addition of coenzyme A (CoA), Luciferyl-CoA was oxidized with advantageous kinetics. After removing the culture medium from the microtiter plate, the cells were lysed by the addition of 100 μΐ lysis buffer (25 mM tris phosphate pH 7.8, 8.2 mM dithiothretol, 2 mM 1,2-diaminocyclohexane-Ν, Ν, Ν ', N'- tetraacetic acid (CDTA), 10% (v / v) glycerin, 1% Triton-X-100). After 15 minutes incubation at room temperature, 10 μΐ of the cell lysate was transferred to a white microtiter plate (Dynatech), suitable for a luminous assay buffer (20 mM Tricine pH 7.8, 1.07 mM (MgCO ₃ ) ₄ Mg (OH) ₂ , 2). , 67 MgSO ₄ , 0.1 mM ethyleneimine tetraacetic acid (EDTA), 33.3 mM dithiothreitol, 270 μΜ of coenzyme A,

470 μΜ locust fly (Photinus pyralis) luciferin,

530 μΜΓΤΡΝδ ₂ ). After one minute, luminescence with a half-life of five minutes was determined for a total time of one second using an EG&G Berthol MicrolLumat LB 96 P.

In this way, the following in vitro data were obtained, with K _D responsible for binding affinities and IC ₅₀ for functional affinity, and pM stands for picomol per liter.

compound K _p (pM) ^IC 50 (PM) cetrorelix 170 (22) 198 (5) Example 1 (acetate salt) not determined 242 (3) Example 2 181 mm (1) 684 (2) Example 3 154 mm (1) 492 (2) Example 6 not determined 221 (2)

() = number of independent experiments

7? 1525-200 /

Claims (15)

PATENT CLAIMS Compounds of formula I Α-Χχχ ¹ -Χχχ ² -Χχχ ³ -Χχχ ⁴ -Χχχ ⁵ -Χχχ ⁶ -Χχχ ⁷ -Χχχ ⁸ -Χχχ ⁹ -Χχχ ¹⁰ -ΝΗ ₂ where they mean A acetyl or 3- (fluorophenyl) propionyl, Xxx ¹ D-Nal (1) or D-Nal (2) Xxx ² -Xxx ³ D-Cpa-D-Pal (3) or single bond, Xxx ⁴ Ser Xxx ⁵ N-Me-Tyr Xxx ⁵ D-Cit, D-Hci or a D-amino acid group of formula (II) (II) wherein n is 3 or 4, wherein R ¹ is a group of formula III ί ~ (CH ₉ ) -CO-NR ² R ³ I ť í (III) wherein p is an integer from 1 to 4, R ² is hydrogen or alkyl and R ³ is unsubstituted or substituted aryl or heteroaryl, or R ¹ is 3-amino-1,2,4-triazole- 5-carbonyl or R ¹ represents a ring of formula (IV) R ⁴ wherein q is 1 or 2, R ⁴ is hydrogen or alkyl, R ⁵ is hydrogen or alkyl, and X is oxygen or sulfur, Xxx ⁷ means Leu or No, Xxx ⁶ Arg or Lys (iPr) Q Xxx Pro a Xxx ¹⁰ Ala or Ser and their salts with pharmaceutically acceptable acids. Compounds according to claim 1, wherein the salt is acetate, trifluoroacetate or embonate. Compounds according to claim 1 or 2, wherein Xxx ⁶ is D- [e-N '- (imidazolidin-2-one-4-yl) -formyl] -Lys, D- (3-amino-1,2,4-triazole-3-carbonyl) -Lys, abbreviated as D- Lys (Atz) or D- [η-N '-4- (4-amidinophenyl) amino-1,4-dioxobutyl] -4-Lys, abbreviated to D-Lys (B). A compound according to claim 1, having the formula Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ 'Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ - Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -nh ₂ . A compound according to claim 1, having the formula Ac-D-Na (2βD-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (Atz) ⁶ -Leu ⁷ - Arg ⁸ - For ⁹ -D-Ala ¹⁰ -NH ₂ . A compound according to claim 1, having the formula Ac-D-Nal (2) ¹ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁹ -D-Lys (B) ⁸ - Leu ⁷ -Lys (iPr) ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂ . . A compound according to claim 1, having the formula Ac-D-Na ( ^2H -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -Lys (B) ⁶ -Leu ⁷ - Arg ⁸ -Pro ⁹ -D 'Ala ¹⁰ -NH ₂ . A compound according to claim 1, having the formula Ac-D-Nal (2) ³ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ - Lys (iPr) ⁸ -Pro ⁹ -Ala ¹⁰ -NH ₂ . A compound according to claim 1, having the formula Ac-D-Nal (2) ³ -D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ - Lys (iPr) ⁸ -Pro ⁹ -Sar ¹⁰ -NH ₂ . 10. A compound according to claim 1, of the formula Ac-D-Nal (2) - ¹ · D-Cpa ² -D-Pal (3) ³ -Ser ⁴ -N-Me-Tyr ⁵ -D-Hci ⁶ -Nle ⁷ -Arg ⁸ -Pro ⁹ -Ser ¹⁰ -NH ₂ . A compound according to claim 1, having the formula 3- (fluorophenyl) propionyl-D-Nal (1) ¹ -Ser ⁴ -N-Me-Tyr ⁵ -D-Lys (Atz) ⁶ -Leu ⁷ -Arg ⁸ -Pro ⁹ -D-Ala ¹⁰ -NH ₂ . A pharmaceutical composition comprising a compound according to any one of claims 1 to 11. 13. A process for the preparation of the compounds of the formula I as claimed in claim 1, characterized in that the fragments of building blocks Xxx ¹¹¹ , provided with suitable protecting groups, in which m is an integer from 1 to 10 and Xxx ¹ is acetylated, are subjected to conventional methods. on solid phase or in solution, then the fragments are attached to the solid phase by coupling the segments and cleaved after the coupling of the compounds of formula I by conventional methods, amidating on the building block Xxx from the solid phase. Use of the substances according to claims 1 to 11 for the manufacture of medicaments for the treatment of hormone-mediated tumors, in particular prostate cancer or breast cancer, as well as benign indications whose treatment requires LH-RH-hormone suppression. A process for the manufacture of medicaments in which the compounds according to claims 1 to 11 are mixed with conventional carriers and excipients and prepared as medicaments.